Vasopressin regulates urea transport by activating two cyclic AMP (cAMP) dependent signaling pathways: protein kinase A and exchange protein activated by cAMP. This results in increases of both the phosphorylation and apical plasma membrane accumulation of the UT-A1 urea transporter. The hypertonicity present in the inner medulla can act independently of vasopressin as a powerful stimulus of urea transport. Hypertonicity stimulates urea transport by increasing both UT-A1 phosphorylation and plasma membrane accumulation. However, the signaling pathway by which this occurs is unknown. We have new preliminary data showing that hypertonicity stimulates urea permeability via protein kinase C (PKC). We also have preliminary data suggesting that PKC1 is the specific PKC isozyme involved since: 1) hypertonicity activates PKC1 in rat inner medullary collecting ducts (IMCDs); and 2) PKC1 knock-out mice have a urine concentrating defect and a reduction in UT-A1 protein abundance. Thus, maximal stimulation of urea transport and UT-A1 activity in the terminal IMCD requires stimulation by both cAMP and PKC1. Our overall goal is to investigate the regulation of UT-A1 by PKC. We will test the hypothesis that PKC1 stimulates UT-A1 function through changes in UT-A1 phosphorylation. Aim 1 will determine the mechanism by which PKC regulates UT-A1 phosphorylation. Aim 2 will determine the site(s) in UT-A1 and the cellular location of phosphorylation by PKC. Aim 3 will determine the interdependence of PKC and PKA in the regulation of UT-A1 membrane accumulation, activity, phosphorylation, and phosphatase-mediated dephosphorylation. Aim 4 will determine the mechanism for the urine concentrating defect in PKC knock-out mice. Our proposed studies are highly significant as they are likely to yield new information on mechanisms underlying dysregulation of water homeostasis. Elucidation of non-cAMP mechanisms for increasing urea transport, which in turn would increase urine concentrating ability, could form the basis for future translational studies of novel therapeutic approaches to congenital nephrogenic diabetes insipidus.